Mitotic Golgi translocation of ERK1c is mediated by PI4KIIIβ/14-3-3γ shuttling complex

Abstract

Golgi fragmentation is a highly regulated process that allows division of the Golgi complex between the two daughter cells. The mitotic reorganization of the Golgi is accompanied by a temporary block in Golgi functioning, as protein transport in and out of the Golgi stops. Our group has previously demonstrated the involvement of the alternatively spliced variants ERK1c and MEK1b (ERK1 is also known as MAPK3, and MEK1 as MAP2K1) in mitotic Golgi fragmentation. We had also found that ERK1c translocates to the Golgi at the G2 to M phase transition, but the molecular mechanism underlying this recruitment remains unknown. In this study, we narrowed the translocation timing to prophase and prometaphase, and elucidated its molecular mechanism. We found that CDK1 phosphorylates Ser343 of ERK1c, thereby allowing the binding of phosphorylated ERK1c to a complex that consists of PI4KIIIβ (also known as PI4KB) and the 14-3-3γ dimer (encoded by YWHAB). The stability of the complex is regulated by protein kinase D (PKD)-mediated phosphorylation of PI4KIIIβ. The complex assembly induces the Golgi shuttling of ERK1c, where it is activated by MEK1b, and induces Golgi fragmentation. Our work shows that protein shuttling to the Golgi is not completely abolished at the G2 to M phase transition, thus integrating several independent Golgiregulating processes into one coherent pathway.

The Great Escape: how phosphatidylinositol 4-kinases and PI4P promote vesicle exit from the Golgi (and drive cancer)

“…PI 4-kinase activity at the TGN also contributes to a minor but functionally important pool of this lipid at the plasma membrane that is involved in agoniststimulated receptor signalling and KCNQ2/3 channel function (63,64). Finally, proteins of the PI4P trafficking machinery can act as binding partners for regulators of the ERK and MAP kinase signalling cascades as has been demonstrated for both OSBP and PI4KIIIβ (16,65,66).…”

High Throughput Analysis of Golgi Structure by Imaging Flow Cytometry

“…These results show that the nocodazole-induced Golgi fragmentation occurs in two phases, first, partial fragmentation, which is observed in the short treatment, and then full fragmentation observed in the long treatment. Although we used a previously reported nocodazole concentration 18, 46, 47 , in other studies a higher concentration of nocodazole (up to 10 μM) was used 48, 49 . To confirm the validity of our results, we repeated the experiments for the 16-hour time point with both 0.3 μM and 10 μM nocodazole.…”

“…3A). Nine hours after the release, the G2/M population was further divided into G2 and the sub-mitotic phases (prophase-telophase) according to cellular and DNA staining morphology, as previously described 46 . Using our method, we then analyzed the percentage of cells with intact, partially and fully fragmented Golgi at each phase.…”

“…The G2/M population was further gated for prophase, prometaphase and mitotic (metaphase-telophase) populations using 2 parameters: the bright detail intensity of Draq5 staining (intensity of localized bright areas, subtracted for the local background), and the area of the 50% highest intensity pixels of the Draq5 staining (defined by the Threshold_50 mask). The different mitotic stages were further defined by the nuclear morphology, as previously described 46 . To quantify the Golgi fragmentation, two features were eventually chosen based on the GM130 staining: Minor Axis Intensity (the intensity weighted narrowest dimension of the ellipse of best fit) and Area (the number of microns squared within a mask).…”

Mitotic inheritance of the Golgi complex and its role in cell division

“…Thus, as the tethering role of the GRASPs can be modulated by phosphorylation (Feinstein and Linstedt, 2008;Sengupta and Linstedt, 2010;Cervigni et al, 2015), we now have the possibility of fine-tuning the ribbon/stack equilibrium by using drugs that inhibit GRASP phosphorylation. It is important to note that the current view of the complex regulatory mechanisms of Golgi organisation is still limited, as other kinases have been shown to be involved in inducing Golgi disassembly, such as PLK3, Vaccinia Related Kinase 1 (VRK1) (Lopez-Sanchez et al, 2009) and ERK1c (Shaul and Seger, 2006;Wortzel et al, 2015), but the definition of their exact role awaits further elucidation. Moreover, systems biology approaches have identified hundreds of potential new regulatory elements of Golgi structure (Chia et al, 2012;Farhan et al, 2010;Farhan, 2015) that remain poorly investigated.…”